In contrast to earlier unpublished observations in this laboratory, it was reported by other workers that the modification of proteins by peroxynitrite leads to the conversion of some amino acid residues to carbonyl derivatives. The discrepancy between our results and those reported earlier has been resolved. It is due to the failure of the other workers to take into account the contribution of light scattering in their spectrophotometric measurements of protein carbonyl content. Nevertheless, in view of the fact that the carbonyl content of proteins is a widely accepted measure of oxidative stress and has been used as a marker of oxidative damage during aging and in many diseases, we investigated further the modification of proteins by peroxynitrite. We confirmed our earlier findings that the oxidation of methionine residues of glutamine synthetase by peroxynitrite is strongly inhibited by physiological concentrations of carbon dioxide, and that the nitration of tyrosine residues is almost completely dependent on the presence of carbon dioxide. We confirmed also that peroxynitrite does not generate significant amounts of protein carbonyl groups at physiological concentrations of hydrogen ion and carbon dioxide. However, the dependence of these reactions on carbon dioxide varies with the concentration of hydrogen ion. These results emphasize the importance of controlling the hydrogen ion concentration and carbon dioxide levels of reaction mixtures in vitro in studies designed to determine the modification of proteins by peroxynitrite. They show further that only marginal levels of protein carbonyl are generated by peroxynitrite under any of the experimental conditions examined. It is therefore unlikely that peroxynitrite contributes significantly to the increase in levels of protein carbonyl groups observed during aging and in various diseases.